The Gram-negative bacterium Francisella tularensis subsp. tularensis, the causative agent of tularemia, is considered a potentially dangerous biological weapon. This is due to the organism's extreme infectivity via the aerosol route and the severity of the human disease it causes. The fastidious nature of the bacterium, the requirement that it be handled using biosafety level three (BSL3) containment, and the paucity of genetic tools have hindered research on this organism. Thus, compared to other pathogens, little is known about F. tularensis subsp. tularensis physiology, genetics, and pathogenesis. Previous studies have used the attenuated live vaccine strain (LVS) derived from F. tularensis subsp. palaearctica, or the rarely pathogenic F. tularensis subsp. novicida. The aim of this proposal is to develop genetic tools for the manipulation of virulent, pathogenic F. tularensis subsp. tularensis. These tools will then be used for the analysis of F. tularensis subsp. tularensis pathogenesis. Furthermore, the ability to genetically manipulate the organism will allow for the development of attenuated vaccine strains that could be administered to populations at risk from F. tularensis infection, either from natural sources or from a biological attack. The further development of Francisella genetics is timely, as the sequencing of the genome of the F. tularensis subsp. tularensis, strain Schu 4 is almost complete.